J Int Adv Otol 2016; 12(3): 237-40 • DOI: 10.5152/iao.2016.3124
Original Article
Single Administration of a Sustained-Release
Formulation of KB-R7785 Inhibits Tympanic Membrane
Regeneration in an Animal Model
Peter Luke Santa Maria, Chloe Santa Maria, Sungwoo Kim, Yunzhi Peter Yang
Department of Otolaryngology, Head and Neck Surgery, Stanford University, California, USA (PLSM)
Department of Ear Sciences, The University of Western Australia, Western Australia, AUS (CSM)
The Ear Science Institute, Western Australia, AUS (CSM)
Department of Orthopedic Surgery, Stanford University, California, USA (SK, YPY)
Department of Materials Science and Engineering, Stanford University, California, USA (YPY)
Department of Bioengineering, Stanford University, California, USA (YPY)
OBJECTIVE: A pressure equalization tube placed within the tympanic membrane is the only clinically available method for inhibiting tympanic
membrane regeneration. Problems associated with this include associated otorrhea, biofilm formation, medial migration of the tube, tube retention, induction of granulation tissue, and a small but significant rate of cholesteatoma. We aimed to demonstrate that a single administration of a
sustained-release polymer formulation of KB-R7785 maintains tympanic membrane perforation for at least 6 months.
MATERIALS and METHODS: Sustained-release KB-R7785 was delivered within a novel polymer hydrogel to 20 mice with bilateral acute tympanic
membrane perforations (a total of 40 perforations). The perforations were monitored at 3-month intervals until 9 months.
RESULTS: At 3 months, 90% of perforations were open (n=36/40). At 6 months, 75% of perforations were open (total n=30/40). At 9 months, 22.5%
of perforations were open (total n=6/40). The majority of tympanic membrane perforations (75%) were open (not healed) beyond 6 months and
close (fully healed) prior to 9 months (77.5%). Once healed, tympanic membranes resembled their normal histological appearance.
CONCLUSION: This study demonstrates that a single administration of a sustained-release polymer formulation of KB-R7785 inhibits tympanic
membrane regeneration for 6–9 months.
KEYWORDS: Tympanic membrane, chronic perforation, tympanic perforation, wound healing
INTRODUCTION
Maintaining a chronic perforation in a tympanic membrane (TM) is part of the current standard of care for a number of middle ear
diseases, including primary Eustachian tube dysfunction, otitis media with effusion, and recurrent acute otitis media [1]. Having a
physical structure, a pressure equalization tube (PET), placed within TM is the only clinically available method for inhibiting TM
regeneration and maintaining an opening. The presence of PET is associated with a number of problems, including tube associated
otorrhea, linked to biofilm formation on the tube, medial migration of the tube, tube retention, induction of granulation tissue,
and a small but significant rate of cholesteatoma [2-4]. There is a need for the ability to maintain a perforation in the TM (through the
inhibition of the normal regenerative mechanisms) that does not require the long-term presence of a physical structure.
KB-R7785 [4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl)-L-phenylglycine-N-methylamide] is a matrix metalloproteinase
(MMP) inhibitor with a particular inhibitory action on MMP-2, MMP-9, disintegrin, and metalloproteinase domain-containing protein 12 (ADAM12) [5]. We have previously demonstrated that KB-R7785 treatment of an acute perforation leads to the inhibition of
tympanic regeneration that persists for at least 3 months [6]. The previously published dosing regimen, for 7 days onto gel foam, is
likely to be difficult and inconvenient, and we aimed to develop a solution that was one-time administration. Our previous experiments have shown the potential of perforations to last for at least 3 months, and for our solution to be a true replacement for PET,
we estimated that the perforations would need to persist for at least 6 months. Because the hypothesis that KB-R7785 inhibits TM
regeneration had already been established, in the present study, we aimed to test the duration for which this inhibition could last
using a sustained-release formulation delivered as a biodegradable polymer.
MATERIALS and METHODS
Stanford University’s Administrative Panel on Laboratory Animal Care approved all animal work (Protocol 27539, approved June
2014). International standards regarding animal care and handling have been followed during the experiments.
Corresponding Address: Peter Luke Santa Maria E-mail: [email protected]
Submitted: 13.10.2016
Revision received: 21.11.2016
Accepted: 22.11.2016
©Copyright 2016 by The European Academy of Otology and Neurotology and The Politzer Society - Available online at www.advancedotology.org
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J Int Adv Otol 2016; 12(3): 237-40
All mice used in this study were 6–10-week-old male CBA/CaJ (15–25
g) mice (Jackson Laboratories, Florida, USA). All recordings by otoscopy and all surgical interventions were performed using inhaled
isoflurane at 3%–4% for induction and 1%–2% for maintenance. The
study design was a prospective cohort one. The determination of animal numbers in this study was performed using STATA version 1.03
(Statacorp LP, Texas, USA), aiming for an α value of 0.05 and a minimum β value of 0.8. STATA version 13.0 (Statacorp LP, Texas, USA) was
used for analysis. In total, 20 mice were used with perforations created bilaterally (a total of 40 perforations). For all statistical analyses,
a Pearson’s Chi-square test of proportions was used. A significance
level of 0.05 was used for the null hypothesis.
KB-R7785
KB-R7785 (4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl)-Lphenylglycine-N-methylamide) was synthesized and obtained from
Stanford University’s chemistry department. It was dissolved in the
prepolymer solution at 10 mM. The KB-R7785 treatment was initiated
for all 40 perforations in 20 mice.
Polymer Construction
Polymer construction has been previously discussed, and the release
profiles of various agents have been characterized [7, 8]. The mass ratio of chitosan to lactide was 8:1. 3.3 mM of sodium metabisulfite,
the cross-linking agent, was added into the prepolymer solution to
form a semi-solid hydrogel within a few minutes. The polymer was
designed to dissolve and release KB-R7785 at 10 nM over a 4-week
period.
Animal Treatment and Inspection
We used a previously published technique to create TM perforations
adapted to mice [6, 9, 10]. In brief, a curved microneedle was used to create a subtotal perforation in the pars tensa of TM [6, 9, 10]. The treatment
was performed via the external auditory canal using a syringe and
27-gauge needle through and onto TM to fill the middle ear and into
the external ear. The total volume delivered was approximately 0.04
mL in each case. The KB-R7785 polymer treatment was initiated for all
40 perforations in 20 mice. The animals were inspected using otosco-
py under general anesthesia at 3-month intervals. Care was taken to
not disturb the TM surface. Otoscopy performed a using the Digital
Macroview (Welch Allyn, New York, USA) otoscope. Perforations were
measured using ImageJ to determine the perforation size as a percentage of the total pars tensa area [11]. A pinhole perforation was defined as a perforation less than 5% of the surface area. All the animals
were sacrificed at 9 months, and TMs were harvested for histology.
Histology
Histology was performed with hematoxylin–eosin staining according to a previously published technique [9].
RESULTS
KB-R7785 delivered via polymer maintains the majority of perforations between 6–9 months
The percentage of healed perforations over time is displayed in Figure 1. At 3 months, 36 perforations were still open (n=36/40, 90%).
At 6 months, further 6 perforations had healed, leaving a total of 30
perforations open (total n= 30/40, 75%), 3 of which healed to pinhole size (total n=27/40, 67.5% larger than pinhole size). At 9 months,
further 21 perforations had healed, leaving a total of 9 perforations
open (total n= 9/40, 22.5%), 3 of which healed to pinhole size (total n=6/40, 15% larger than pinhole size). There was no significant
difference between the proportion of perforations open at 3 and 6
months (χ2=1.02, p=0.31); however, there were significantly more
perforations open at 6 months than at 9 months (χ2=26.8, p<0.001).
Healed TMs after KB-R7785 treatment resemble normal TMs
All healed TMs (Figure 2a) after treatment resembled normal TM histology with an inner mucosal layer, a middle connective tissue layer,
and an outer (darker) thin keratinocyte layer. In comparison, all persisting chronic perforations had thickened and disorganized connective tissue layers and thickened keratinocyte layers reaching over the
perforation edge to lie adjacent to the mucosal layer (Figure 2b). No
inflammation or mucosal thickening was identified in the middle ear
mucosa of the specimens at 9 months.
DISCUSSION
Figure 1. Tympanic membrane perforation open after KB treatment over
time. Figure 1 demonstrates the percentage of tympanic membrane perforations (n=40) open (not healed) after treatment with KB-R7785 at 3-, 6-,
and 9-month inspections, shown in the solid line (% TOTAL OPEN). The majority (75%) were open (not healed) at least for 6 months and close prior to
9 months (77.5%). A small percentage had healed to a small pinhole (≤5%
of the total pars tensa surface area) size at each time point, as shown in the
dashed line (% OPEN>pinhole size).
238
Animal Model Limitations
While the mice were alive, it was easy to determine the presence
or absence of a perforation via transcanal otoscopy, but it was not
possible to determine the true size of the perforations because
there was often covering debris or discharge. The angle of TM in
the narrow mouse ear canal makes it difficult for a total inspection
of the whole pars tensa surface in vivo. This required removal of
the debris, which would injure TM and artificially make the perforation seem larger. Therefore, we were unable to comment in vivo on
how the perforation was changing or possibly reducing in size accurately. The other limitation was the potential for bias in reporting
because only a single observer could report on the perforations.
Getting a clearer view of the perforations via the removal of debris
for in vivo photography would have disturbed the perforation and
introduced another confounding factor in the healing. The disadvantage of KB-R7785 in its current delivery form and this animal
model is that it is difficult to maintain a patent perforation in the
early days because the polymer itself obstructs the perforation
Santa Maria et al. KB-R7785 Inhibits Tympanic Membrane Regeneration
a
b
Figure 2. a, b. Tympanic membranes at 9 months following KB-R7785. Figure 2 demonstrates the tympanic membrane after treatment KB-R7785 at 9 months (a)
in a healed perforation (b) in a persisting chronic perforation. The arrow identifies the perforation margin showing a thicker keratinocyte and connective tissue
layer. Scale bar=5µm (Magnification 40×).
opening. If translated directly, this would prevent the early release
of middle ear fluid and ventilation of the middle ear, a benefit of
PETs. The polymer also results in temporary hearing loss, which resolves after the polymer dissolves [6]. For this purpose, the delivery
vehicle would need to be modified to be delivered adjacent to the
perforation opening without obstructing it. This would be difficult
to test in a small-sized TM, as observed in the mouse, and would
require a larger animal model for proof of concept.
KB-R7785 acts via inhibition of epidermal growth factor receptor
ligand shedding
KB-R7785 administration is thought to inhibit TM wound healing
via the inhibition of heparin-binding epidermal growth factor-like
growth factor (HB-EGF)-mediated epidermal growth factor receptor
(EGF-R) ectodomain ligand shedding [6, 12-15]. This migration and proliferation of keratinocytes from progenitor cells located at the attachment of TM to the malleus handle play a crucial role in TM wound
healing [9]. The first 7 days appear to be critical in TM wound healing
to determine whether a perforation heals or becomes chronic [6, 9].
KB-R7785 also has been identified as having a potential role in modulating proliferation in the gastric mucosa, vascular smooth muscle,
tumor cell migration, cerebral ischemia, and apoptosis in the bone
marrow [5, 16-20].
KB-R7785 delivered via polymer has potential to replace PETs
Previously, it has been demonstrated that chronic perforations can
be created using daily-repeated KB-R7785 (10nM) administration
over 1 week onto gel foam into an acute TM perforation. In the present study, a single administration of KB-R7785 via a novel delivery
vehicle enabled perforations to be maintained for at least 6 months.
This study also showed that this single administration does not produce a persisting perforation, in the majority, beyond 9 months.
Given this timing of chronic perforation, this treatment has the potential to replace the physical obstruction method of PETs if further
developed into a suitable structure that allows ventilation in the
early period. Once TM is healed following KB-R7785 inhibition, the
histology takes the appearance of a normal TM. This is important for
implications about the potential development of tympanosclerosis
and other histological changes observed in TM following subacute
or chronic perforation.
KB-R7785 delivered via polymer requires further development
before use in human ear
Although the drug release profile of other agents has been investigated in this current delivery system, the current release profile of
KB-R7785 needs to be defined. The current proposed polymer has
already been shown to be nonototoxic [6]. Other studies delivering
KB-R7785 parenterally in rodents have not shown systemic toxicity;
however, these were not the primary endpoints measured in these
studies [5]. Despite there being no middle ear histological changes
detected after tympanic healing, further toxicity studies, specifically
for KB-R7785, are needed before it is deemed safe to progress toward
human clinical trials.
CONCLUSION
This study demonstrated that a single administration of a sustained-release polymer formulation of KB-R7785 inhibits TM regeneration between 6–9 months.
Ethics Committee Approval: Ethics committee approval was received for this
study from the ethics committee of Stanford University.
Informed Consent: N/A.
Peer-review: Externally peer-reviewed.
Author Contributions: Concept - P.L.S.M., S.K., Y.P.Y.; Design - P.L.S.M., C.S.M.,
S.K., Y.P.Y.; Supervision - P.L.S.M., C.S.M., S.K., Y.P.Y.; Resources - P.L.S.M., Y.P.Y.;
Materials - P.L.S.M., S.K., Y.P.Y.; Data Collection and/or Processing - P.L.S.M.,
C.S.M.; Analysis and/or Interpretation - P.L.S.M., C.S.M., S.K., Y.P.Y.; Literature
Search - P.L.S.M., S.K.; Writing Manuscript - P.L.S.M., C.S.M., S.K., Y.P.Y.; Critical
Review - P.L.S.M., C.S.M., S.K., Y.P.Y.
Conflict of Interest: Filed patents: U.S. Non Provisional No. 61/823,749, April
2014: P Santa Maria, S Kim, Y Yang - U.S. Non Provisional No. 61/810,101, April
2014: S Kim, Y Yang. Founding stock in Auration biotech: P Santa Maria.
Financial Disclosure: Garnett Passe and Rodney Williams Memorial Foundation. Stanford’s SPARK. Stanford Child Health Research Institute. NIH
R01AR057837 (NIAMS, YY), NIH R01DE021468 (NIDCR, YY), DOD W81XWH-10-1-0966 (PRORP, YY), and Wallace H. Coulter Foundation (YY).
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J Int Adv Otol 2016; 12(3): 237-40
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
240
Rettig E, Tunkel DE. Contemporary concepts in management of acute
otitis media in children. Otolaryngol Clin North Am 2014; 47: 651-72.
Barakate M, Beckenham E, Curotta J, da Cruz M. Bacterial biofilm adherence to middle-ear ventilation tubes: scanning electron micrograph images and literature review. J Laryngol Otol 2007; 121: 993-7.
Groblewski JC, Harley EH. Medial migration of tympanostomy tubes:
an overlooked complication. Int J Pediatr Otorhinolaryngol 2006; 70:
1707-14.
Kay DJ, Nelson M, Rosenfeld RM. Meta-analysis of tympanostomy tube
sequelae. Otolaryngol Head Neck Surg 2001; 124: 374-80.
Jiang X, Namura S, Nagata I. Matrix metalloproteinase inhibitor KBR7785 attenuates brain damage resulting from permanent focal cerebral
ischemia in mice. Neurosci Lett 2001; 305: 41-4.
Santa Maria PL, Varsak KY, Kim S, Yang YP. Heparin Binding – Epidermal
Growth Factor Like Growth Factor for Regeneration of Chronic Tympanic
Membrane Perforations in Mice. Tissue Eng Part A 2015; 21: 1483-94.
Kim S, Kang Y, Krueger CA, Sen M, Holcomb JB, Chen D, et al. Sequential delivery of BMP-2 and IGF-1 using a chitosan gel with gelatin microspheres enhances early osteoblastic differentiation. Acta Biomater 2012;
8: 1768-77.
Kim S, Tsao H, Kang Y, Young DA, Sen M, Wenke JC, et al. In vitro evaluation of an injectable chitosan gel for sustained local delivery of BMP-2 for
osteoblastic differentiation. J Biomed Mater Res B Appl Biomater 2011;
99: 380-90.
Santa Maria PL, Redmond SL, Atlas MD, Ghassemifar R. Histology of the
healing tympanic membrane following perforation in rats. Laryngoscope 2010; 120: 2061-70.
Varsak KY, Santa Maria PL. Mouse Model of Experimental Eustachian
Tube Occlusion: A Surgical Technique. Acta Oto-Laryngologica 2015;
136: 12-7.
Rasband WS. ImageJ Software, National Institutes of Health, Bethesda,
MD, USA 1997-2004. Available from: http://rsb.info.nih.gov/ij/.
12. Santa Maria PL, Redmond SL, Atlas MD, Ghassemifar R. The role of epidermal growth factor in the healing tympanic membrane following perforation in rats. J Mol Histol 2010; 41: 309-14.
13. Santa Maria PL, Weierich K, Kim S, Yang YP. Heparin Binding Epidermal
Growth Factor-Like Growth Factor Heals Chronic Tympanic Membrane
Perforations With Advantage Over Fibroblast Growth Factor 2 and
Epidermal Growth Factor in an Animal Model. Otol Neurotol 2015; 36:
1279-83.
14. Tokumaru S, Higashiyama S, Endo T, Nakagawa T, Miyagawa JI, Yamamori K, et al. Ectodomain shedding of epidermal growth factor receptor
ligands is required for keratinocyte migration in cutaneous wound healing. J Cell Biol 2000; 151: 209-20.
15. Shirakata Y, Kimura R, Nanba D, Iwamoto R, Tokumaru S, Morimoto C, et
al. Heparin-binding EGF-like growth factor accelerates keratinocyte migration and skin wound healing. J Cell Sci 2005; 118: 2363-70.
16. Joh T, Kataoka H, Tanida S, Watanabe K, Ohshima T, Sasaki M, et al. Helicobacter pylori-stimulated interleukin-8 (IL-8) promotes cell proliferation
through transactivation of epidermal growth factor receptor (EGFR) by
disintegrin and metalloproteinase (ADAM) activation. Dig Dis Sci 2005;
50: 2081-9.
17. Zhang H, Chalothorn D, Jackson LF, Lee DC, Faber JE. Transactivation
of epidermal growth factor receptor mediates catecholamine-induced
growth of vascular smooth muscle. Circ Res 2004; 95: 989-97.
18. Arimura K, Arima N, Matsushita K, Ohtsubo H, Fujiwara H, Kukita T, et al.
Matrix metalloproteinase inhibitor reduces apoptosis induction of bone
marrow cells in MDS-RA. Eur J Haematol 2004; 73: 17-24.
19. Murai T, Miyazaki Y, Nishinakamura H, Sugahara KN, Miyauchi T, Sako Y,
et al. Engagement of CD44 promotes Rac activation and CD44 cleavage
during tumor cell migration. J Biol Chem 2004; 279: 4541-50.
20. Madarame J, Higashiyama S, Kiyota H, Madachi A, Toki F, Shimomura
T, et al. Transactivation of epidermal growth factor receptor after heparin-binding epidermal growth factor-like growth factor shedding in
the migration of prostate cancer cells promoted by bombesin. Prostate
2003; 57: 187-95.